Electrophoresis buffer for faster migration, improved resolution and extended shelf-life

ABSTRACT

There is provided an electrolyte solution for extending shelf life, and/or accelerating or improving resolution or improving transfer efficacy for blot applications, or accelerating and improving resolution, or accelerating and improving transfer efficacy of gel electrophoresis and containing Tris(hydroxymethyl)aminomethane (TRIS), at least one zwitterion, and water. The electrolyte solution may be used in buffer systems for gel electrophoresis and the preparation of gels for gel electrophoresis, such as Western blot.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a U.S. National Phase Patent Application of PCT/CA2011/000500,filed Apr. 21, 2011, which claims the benefit under 35 USC 119(e) toU.S. Provisional Application No. 61/440,096, filed Feb. 7, 2011, U.S.Provisional Application No. 61/431,757, filed Jan. 11, 2011, U.S.Provisional Application No. 61/373,315, filed Aug. 13, 2010, and U.S.Provisional Application No. 61/328,208, filed Apr. 27, 2010, each ofwhich is hereby incorporated by reference in the present disclosure inits entirety.

BACKGROUND

(a) Field

The subject matter disclosed generally relates to gel electrophoresis.More specifically, the subject matter disclosed relates to anelectrolyte solution for accelerating or improving resolution, oraccelerating and improving resolution of gel electrophoresis andcontaining Tris(hydroxymethyl)aminomethane (TRIS), at least onezwitterion, and water.

(b) Related Prior Art

Gel electrophoresis is a common procedure for the separation ofbiological molecules, such as deoxyribonucleic acid (DNA), ribonucleicacid (RNA), polypeptides and proteins. In gel electrophoresis, themolecules are separated into bands according to the rate at which animposed electric field causes them to migrate through a filtering gel.

The basic apparatus used in this technique consists of a gel enclosed ina glass tube, sandwiched as a slab between glass or plastic plates, orpoured in a plastic tray. The gel has an open molecular networkstructure, defining pores which are saturated with an electricallyconductive buffered solution. These pores through the gel are largeenough to admit passage of the migrating macromolecules.

The gel is placed in a chamber in contact with buffer solutions whichmake electrical contact between the gel and the cathode or anode of anelectrical power supply. A sample containing the macromolecules and atracking dye is placed on top of the gel. An electric potential isapplied to the gel causing the sample macromolecules and tracking dye tomigrate toward the bottom of the gel. The electrophoresis is halted justbefore the tracking dye reaches the end of the gel.

The most common buffer system employed for the separation of proteins isthe Laemmli buffer system consists of 0.375 M tris (hydroxymethyl)amino-methane (Tris), titrated to pH 8.8. with HCl, in theseparating gel. The stacking gel consists of 0.125 M Tris, titrated topH 6.8. The anode and cathode running buffers contain 0.024 M Tris,0.192 M glycine, 0.1% SDS. Many different gel separation materials havebeen disclosed, with different compositions, pH characteristics, voltagerequirements, etc. The goal of most of the recent innovations in thefield has been to provide an electrophoresis gel which can be used toperform a faster, more accurate, more stable, or therefore moreversatile electrophoresis.

A number of different gel buffer systems have been proposed for use ator around neutral pH that do not involve the use of the Tris-HCl Glycinebuffer system of Laemmli.

For example, U.S. Pat. No. 6,096,182 to Updyke et al. discloses anelectrophoresis gel at a neutral pH. The advantage of producing such agel is that the gel system is stable, with reduced reactivity andincreased shelf life.

U.S. Pat. No. 5,464,517 to Hjerten et al. discloses an electrophoresisbuffer which has a high buffering capacity and low electricalconductivity. The advantage of this type of buffer, particularly incapillary electrophoresis, is that it allows the separation to beperformed at a higher voltage and consequently more quickly.

A majority of innovations have focused on improving electrophoresis byproposing new recipes for the gel buffer.

Therefore, there is a need for reagents that will improve the speed atwhich electrophoresis can be performed, improve gel resolution, as wellas increase the shelf life of gels.

SUMMARY

In a first embodiment there is disclosed an electrolyte solution foraccelerating, or improving resolution, or improving transfer efficacyfor blot applications, including Western blot for example, oraccelerating and improving resolution, or accelerating and improvingWestern transfer efficacy of gel electrophoresis containing:

-   -   Tris(hydroxymethyl)aminomethane (TRIS);    -   at least one zwitterion that may be chosen from        2-amino-2-methyl-1,3-propanediol (AMPD),        N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),        N-Glycylglycine (Gly-Gly),        4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or        HEPPS), and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid        (CAPSO),        N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic        acid (AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),        2-(cyclohexylamino)ethanesulfonic acid (CHES),        3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid        (DIPSO), N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid)        (HEPBS), 4-(N-Morpholino)butanesulfonic acid (MOBS),        3-N-Morpholino propanesulfonic acid (POPSO),        N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),        N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),        (2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic        acid) (TES)), (2-[2-acetamino]-2-aminoethanesulphonic acid)        (ACES), (1,4-piperazinediethanesulphonic acid) (PIPES),        (31[N-morpholino]propancsulphonic acid) (MOPS), and        piperazine-N,N′-bis(3-propanesulfonic Acid) (PIPPS); and    -   water.

The TRIS component of the above electrolyte solution may be selectedfrom the group consisting of: Tris-HCl Glycine, Tris-Glycine,MOPS-Bis-Tris, EPPS-Tris-SDS, and the like.

The zwitterion may be chosen fromN,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),2-(cyclohexylamino)ethanesulfonic acid (CHES), N-Glycylglycine(Gly-Gly), 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS orHEPPS), (2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonicacid) (TES)), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),(1,4-piperazinediethanesulphonic acid) (PIPES),(3-[N-morpholino]propancsulphonic acid) (MOPS), andN-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) foraccelerating gel electrophoresis.

The zwitterion may be chosen fromN-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),(1,4-piperazinediethanesulphonic acid) (PIPES),(3-[N-morpholino]propancsulphonic acid) (MOPS), andN-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) forimproving resolution of gel electrophoresis.

The zwitterion may be chosen from N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS)),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), (2-[2-acetamino-1-2-aminoethanesulphonic acid) (ACES),(1,4-piperazinediethanesulphonic acid) (PIPES),(3-[N-morpholino]propancsulphonic acid) (MOPS), andN-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) for,accelerating and improving resolution of gel electrophoresis

The zwitterion may be chosen from N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES), and3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) foraccelerating and improving Western transfer efficacy of gelelectrophoresis.

The zwitterions may be chosen from N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES), and3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) can also beused in combination with one or more other zwitterions from the group2-amino-2-methyl-1,3-propanediol (AMPD),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),2-(cyclohexylamino)ethanesulfonic acid (CHES),3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS),4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholinopropanesulfonic acid (POPSO),N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesul phonic acid)(TES) and piperazine-N,N′-bis(3-propanesulfonic Acid) (PIPPS) and keepits accelerating efficacy in electrophoresis separation and Westerntransfer as well as providing improved separation resolution.

The zwitterions for improving resolution may be4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS).

The electrolyte solution may have a pH from about 8.0 to about 8.8.

The electrolyte solution may comprise methanol, up to 20%.

The electrolyte solution may comprise sodium dodecyl sulphate (SDS) orlithium dodecyl sulfate (LDS), up to 5%.

The electrolyte solution may comprise a chelating agent having the name:ethylenediaminetetraacetate (EDTA), ethylene glycol tetraacetic acid(EGTA), trisodium nitrilotriacetate, hydroxyethyl ethylenediaminetrisodium acetate (trisodium HEDTA), diethylene triamino pentasodiumacetate or uramil disodium acetate.

The concentration of Tris(hydroxymethyl)aminomethane (TRIS) may be fromabout 10 mM to about 500 mM, from about 50 mM to about 150 mM, or fromabout 50 mM to about 250 mM, or may be 150 mM.

The concentration of the zwitterion may be from about 1 mM to 500 mM,from about 10 mM to about 500 mM, or from about 25 mM to about 50 mM, orfrom about 50 mM to about 100 mM, or may be 50 mM or may be 100 mM.

The concentration of sodium dodecyl sulphate (SDS) may be 0.5% (wt/vol)or less, or may be 0.1% (wt/vol) or less, or may be 0.1% (wt/vol).

The concentration of ethylenediaminetetraacetate (EDTA) may be 0.5%(wt/vol) or less, or may be 0.05% (wt/vol) or less or may be 0.03%(wt/vol).

The zwitterions may be a combination of4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),(2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),(1,4-piperazinediethanesulphonic acid) (PIPES), and(3-[N-morpholino]propancsulphonic acid) (MOPS).

In a second embodiment, there is disclosed an electrophoresis gelcomprising the electrolyte solution according to the present invention.The gel may be an acrylamide gel, and the concentration of acrylamidemay be from about 4% (wt/vol) to about 25% (wt/vol). The gel may be anagarose gel, and the concentration of agarose may be from about 0.5%(wt/vol) to about 3% (wt/vol). The gel may be an acrylamide and agarosegel, and the concentration of acrylamide may be from about less than 1%(wt/vol) to about 25% (wt/vol) and the concentration of agarose may befrom about 0.5% (wt/vol) to about 3% (wt/vol). The electrophoresis gelmay comprise sodium dodecyl sulphate (SDS).

The zwitterion may be chosen from 2-amino-2methyl-1,3-propanediol(AMPD), N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonicacid (AMPSO), N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),2-(cyclohexylamino)ethanesulfonic acid (CHES),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS)and 3-N-Morpholino propanesulfonic acid (POPSO) for improving shelf lifeof the gel.

The pH of the gel may be from about 8.0 to about 8.8.

In yet another embodiment, there is disclosed a method of acceleratingor improving the resolution, or accelerating and improving theresolution of the electrophoretic separation of at least one samplecomprising the step of applying a voltage to an electrolyte solutionaccording to the present invention, in contact with an electrophoresisgel containing the at least one sample therein.

The electrophoresis gel is a gel according to the present invention.

In yet another embodiment, there is disclosed a method of acceleratingor improving the resolution of, or improving transfer efficacy, oraccelerating and improving the resolution, or accelerating and improvingtransfer efficacy of the electrophoretic separation of at least onesample comprising the step of applying a voltage to an electrolytesolution adapted to perform gel electrophoresis in contact with anelectrophoresis gel according to the present invention containing the atleast one sample therein.

In yet another embodiment, there is disclosed a method for improvingresolution of an electrophoretic separation of at least one samplecomprising adding at least one zwitterion chosen from2-amino-2methyl-1,3-propanediol (AMPD),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS), and3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),2-(cyclohexylamino)ethanesulfonic acid (CHES),3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS),4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholinopropanesulfonic acid (POPSO),N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),(1,4-piperazinediethanesulphonic acid) (PIPES),(3-[N-morpholino]propancsulphonic acid) (MOPS), andpiperazine-N,N′-bis(3-propanesulfonic Acid) (PIPPS) to anelectrophoresis buffer or to an electrophoresis gel, or to both anelectrophoresis buffer and an electrophoresis gel. The zwitterion may be4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS).

In yet another embodiment, there is disclosed a use of an electrolytesolution according to the present invention for accelerating gelelectrophoresis.

In yet another embodiment, there is disclosed a use of an electrolytesolution according to the present invention for improving resolution ofgel electrophoresis.

In yet another embodiment, there is disclosed a use of an electrolytesolution according to the present invention for accelerating andimproving resolution of gel electrophoresis.

In yet another embodiment, there is disclosed a use of a gel accordingto the present invention for accelerating or improving resolution, oraccelerating and improving resolution of gel electrophoresis.

In yet another embodiment, there is disclosed a use of an electrolytesolution according to the present invention for the preparation of anelectrophoresis gel, wherein the zwitterions is a compound of formula:2-amino-2methyl-1,3-ropanediol (AMPD),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),2-(cyclohexylamino)ethanesulfonic acid (CHES),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) or3-N-Morpholino propanesulfonic acid (POPSO) for improving shelf life ofa gel.

The following terms are defined below.

The term “improved resolution” is intended to mean a better resolutionwhich allows separation of sharper or narrower bands of molecules,distanced or spaced apart from each other as opposed to other means ofseparation which have broader or thicker bands. This facilitatesphysical separation or molecular weight identification of the differentmolecules that make up these bands over the entire range of molecularweight.

Features and advantages of the subject matter hereof will become moreapparent in light of the following detailed description of selectedembodiments, as illustrated in the accompanying figures. As will berealized, the subject matter disclosed and claimed is capable ofmodifications in various respects, all without departing from the scopeof the claims. Accordingly, the drawings and the description are to beregarded as illustrative in nature, and not as restrictive and the fullscope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates polyacrylamide gels electrophoresed in electrolytesolutions according to embodiments of the present invention and comparedto a gel electrophoresed on Tris-glycine-SDS (baseline) solution.

FIG. 2 illustrates polyacrylamide gels electrophoresed in electrolytesolution according an embodiment of the present invention.

FIG. 3 illustrates polyacrylamide gels prepared with electrolytesolutions according to embodiments of the present invention, and kept at4° C. to age during 360 days. Each month, gels were run to measurechanges in resolution, migration path and migration speed.

FIG. 4 illustrates membranes comprising prestained protein marker thathave been transferred from polyacrylamide gels by (A) wet transfer, (B)semi-dry transfer, (C) asymmetric buffer for a “dry” transfer, and (D) acontrol semi-dry transfer using the classic Towbin buffer, run 275 mAfor 15 minutes. The transfer in D is not as efficient. The runprestained protein markers range from 6 KDa to 245 KDa.

FIG. 5 illustrates a prestained protein marker that have beentransferred from polyacrylamide gels in the eStain™ apparatus using a 5×solution of an EPPS (or HEPPS) containing buffer according to thepresent invention.

FIG. 6 (A-L) illustrates a comparison of the migration resolution ofMOPS based gels in an electrolyte solutions according to the presentinvention vs. the regular running buffer for the MOPS based gels.

FIG. 7 (A-C) illustrates a comparison of the migration resolution of 10%acrylamide Tris-glycine based gel (A), 4-20% acrylamide Tris-glycinebased gel (B), and 10% acrylamide EPPS based gel, in an electrolytesolutions according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have now surprisingly found that selectingspecific zwitterions to prepare an electrolyte solution for use in gelelectrophoresis can result in unexpected increase in electrophoresisspeed, improvement in gel resolution, or both. These improvements may beobserved when the electrolyte solutions are used as running buffer (alsoreferred to as “reservoir” buffer) for the electrophoresis apparatus, aswell as when used in as buffer system for the preparation of theelectrophoresis gels (i.e. in the gel). Furthermore, when used in thepreparation of electrophoresis gels, some of these zwitterions will alsoincrease in the shelf life of gels prepared using these electrolytesolutions.

In embodiments there are disclosed an electrolyte solution forperforming gel electrophoresis. The electrolyte contains specificcomponents.

Zwitterions

A zwitterion is a chemical compound that carries a total net charge of 0and is thus electrically neutral, but carries formal charges ondifferent atoms. Zwitterions are polar and are usually verywater-soluble, but poorly soluble in most organic solvents. Zwitterionswill exist mostly as zwitterions in a certain range of pH. The pH atwhich the average charge is zero is known as the molecule's isoelectricpoint.

The zwitterions of interest in the present invention belong to thecategory commonly referred to as biological buffers, which are buffersthat are commonly used as buffering agents in biological laboratories.Examples of biological buffers that can be cited are those known asbis-TRIS (2-bis[2-hydroxyethyl]amino-2-hydroxymethyl-1,3-propanediol),ADA (N-[2-acetamido]-2-iminodiacetic acid), ACES(2-[2-acetamino[-2-aminoethanesulphonic acid), PIPES(1,4-piperazinediethanesulphonic acid), MOPSO(3-[N-morpholino]-2-hydroxypropanesulphonic acid), bis-TRIS PROPANE(1,3-bis[tris(hydroxymethyl)methylaminopropane]), BES(N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid), MOPS(3-[N-morpholino]propancsulphonic acid), TES(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid),HEPES (N-[2-hydroxyethyl]piperazine-N′-(2-ethanesulphonic)acid), DIPSO(3-N,N-bis[2-hydroxyethyl]amino-2-hydroxypropanesulphonic acid), MOBS(4-N-morpholinobutanesulphouic acid), TAPSO(3[N-tris-hydroxymethyl-methylamino]-2-hydroxypropanesulphonic acid),TRIS (2-amino-2-[hydroxymethyl]-1,3-propanediol),HEPPSO(N12-hydroxyethyl]piperazine-N′42-hydroxypropanesulphonic]acid),POPSO (piperazie-N,N′-bis[2-hydroxypropanesulphonic]acid), TEA(triethanolamine), EPPS (or HEPPS)(N-[2-hydroxyethyl]-piperazine-N′-[3-propanesulphonic]acid), TRICINE(N-tris[hydroxymethyl]methylglycine), GLY-GLY (diglycine), BICINE(N,N-bis[2-hydroxyethyl]glycine), HEPBS(N-[2-hydroxyethyl]piperazine-N′-[4-butanesulphonic]acid),TAPS(N-tris[hydroxymethyl]methyl-3-aminopropanesulphonic acid), AMPD(2-amino-2-methyl-1,3-propanediol), TABS(N-tris[hydroxymethyl]methyl-4-aminobutanesulphonic acid), AMPSO(3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulphonic acid),CHES (2-(N-cyclohexylamino)ethanesulphonic acid), CAPSO(3-[cyclohexylamino]-2-hydroxy-1-propanesulphonic acid), AMP(2-amino-2-methyl-1-propanol), CAPS(3-cyclohexylamino-1-propanesulphonic acid), and CABS(4-[cyclohexylamino]-1-butanesulphonic acid).

Although the biological buffering property of these zwitterions has beenrecognized, the capacity of a select group of these zwitterions topositively impact the performance of gel electrophoresis, and the shelflife of gels for gel electrophoresis was not.

Preferably the zwitterions include ACES(2-[2-acetamino[-2-aminoethanesulphonic acid), PIPES(1,4-piperazinediethanesulphonic acid), MOPS(3-[N-morpholino]propancsulphonic acid),2-amino-2-methyl-1,3-propanediol (AMPD), N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS), and3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),2-(cyclohexylamino)ethanesulfonic acid (CHES),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), 3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid(DIPSO), 4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS orHEPPS), N-Glycylglycine (Gly-Gly),N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS),4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholinopropanesulfonic acid (POPSO),N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) andpiperazine-N,N′-bis(3-propanesulfonic Acid) (PIPPS). These zwitterionssignificantly improve the speed at which electrophoresis may beperformed, improve gel resolution, or both. The zwitterions may improveresolution by improving the definition (or sharpness), by providingthinner or narrow bands) of the molecules separated, by improving theseparation between the molecules (i.e. the distance between theindividual bands, or by both improving the definition (or sharpness) andseparation of the molecules. Also, when used for the preparation ofgels, increase in the shelf life of gels prepared using theseelectrolyte solutions.

The ranges of concentration over which these zwitterions may be used forthe preparation of electrolyte solutions according to the presentinvention, for accelerating gel electrophoresis and/or improving gelresolution discussed herein are from about 1 mM to about 500 mM, or fromabout 10 mM to about 500 mM, or from about 25 mM to about 50 mM, or fromabout 25 mM to about 100 mM, or from about 10 mM to about 100 mM, orfrom about 1 mM to about 100 mM, or from about 1 mM to about 75 mM, orfrom about 10 mM to about 75 mM, or from about 1 mM to about 10 mM, orfrom about 1 mM to about 50 mM or from about 10 mM to about 50 mM.

The ranges of concentration over which these zwitterions may be used forimproving the shelf life of gels prepared according to the presentinvention are from about 1 mM to about 500 mM, or from about 10 mM toabout 500 mM, or from about 10 mM to about 100 mM, or from about 1 mM toabout 100 mM, or from about 1 mM to about 75 mM, or from about 10 mM toabout 75 mM, or from about 1 mM to about 10 mM, or from about 1 mM toabout 50 mM or from about or from about 10 mM to about 50 mM or, fromabout 25 mM to about 375 mM, and preferably, at 100 mM.

Preferably, the zwitterions to accelerate gel electrophoresis (ascompared to the classical Tris-Glycine-SDS at pH 8.3 of Laemmli) are2-amino-2methyl-1,3-propanediol (AMPD),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),2-(cyclohexylamino)ethanesulfonic acid (CHES), N-Glycylglycine(Gly-Gly), 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS orHEPPS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),ACES (2-[2-acetamino[-2-aminoethanesulphonic acid), PIPES(1,4-piperazinediethanesulphonic acid), MOPS(3-[N-morpholino]propancsulphonic acid), and(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES). When using the above zwitterions in the electrolyte solutionsaccording to the present invention, for running and/or preparing anelectrophoresis gel, the migration speed of gels may be increase byabout 30% or more. The zwitterions may be used alone or in combination.

Optimal results with 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid(EPPS or HEPPS) are obtained at pH of about pH 8.0 to 8.8.

Preferably the zwitterions to improve the resolution of gels duringelectrophoresis (as compared to the classical Tris-Glycine-SDS at pH 8.3of Laemmli) areN-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-N-Morpholino propanesulfonic acid (POPSO) ACES(2-[2-acetamino[-2-aminoethanesulphonic acid), PIPES(1,4-piperazinediethanesulphonic acid), MOPS(3-[N-morpholino]propancsulphonic acid), andN-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS). Whenusing the above zwitterions in the electrolyte solutions according tothe present invention, for running and/or preparing an electrophoresisgel, the resolution of the gels may be increase by 40% or more. Mostpreferably the zwitterion to improve the resolution of gels duringelectrophoresis is 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid(EPPS or HEPPS). The resolution may be improved by improving thedefinition (or sharpness), by providing thinner or narrow bands) of themolecules separated, by improving the separation between the molecules(i.e. the distance between the individual bands, or by both improvingthe definition (or sharpness) and separation of the molecules. Accordingto one embodiment of the present invention, the improvement inresolution may result in a normal, continuous acrylamide concentrationgel showing characteristic similar to a gradient concentration ofacrylamide gel. These gradient-like improvements in resolution offerbetter and greater separation between bands of molecules, whilemaintaining or improving sharpness of the protein bands.

Preferably, the zwitterions to accelerate gel electrophoresis and toimprove the resolution of gels during electrophoresis (as compared tothe classical Tris-Glycine-SDS at pH 8.3 of Laemmli) areN,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),N-Glycylglycine (Gly-Gly),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ACES(2-[2-acetamino[-2-aminoethanesulphonic acid), PIPES(1,4-piperazinediethanesulphonic acid), MOPS(3-[N-morpholino]propancsulphonic acid), and4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS).When using the above zwitterions in the electrolyte solutions accordingto the present invention, for running and/or preparing anelectrophoresis gel, the resolution of the gels may be increase by 40%or more and the migration speed of gels may be increase by about 30% ormore. Most preferably the zwitterion to accelerate gel electrophoresisand to improve the resolution of gels during electrophoresis is4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS). Theresolution may be improved by improving the definition (or sharpness),by providing thinner or narrow bands) of the molecules separated, byimproving the separation between the molecules (i.e. the distancebetween the individual bands, or by both improving the definition (orsharpness) and separation of the molecules.

Preferably the zwitterions to improve the shelf life of gels (ascompared to the classical Tris-Glycine-SDS gels of Laemmli) are2-amino-2methyl-1,3-propanediol (AMPD),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),2-(cyclohexylamino)ethanesulfonic acid (CHES), and 3-N-Morpholinopropanesulfonic acid (POPSO).

Optimal results with 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid(EPPS or HEPPS) are obtained at pH of about pH 8.0 to 8.8.

Tris(hydroxymethyl)aminomethane Base (TRIS)

Tris is an abbreviation of the organic compound known astris(hydroxymethyl)aminomethane, with the formula (HOCH₂)₃CNH₂. Tris isextensively used in biochemistry and molecular biology. In biochemistry,Tris is widely used as a component of buffer solutions, such as in TAEand TBE buffer, especially for solutions of nucleic acids.

Tris also allows the pH of the electrolyte solution of the presentinvention to be set towards more basic pH values. Tris also has apositive impact on the migration speed in the preferred concentrationranges.

The ranges of concentration over which Tris base may be used for thepreparation of electrolyte solutions according to the present invention,for accelerating gel electrophoresis and improving gel resolution asdiscussed herein are from about 10 mM to about 500 mM. The Trisconcentration has a direct impact on the pH of the solution. Anincreasing amount of Tris increases the pH. This allow fine adjustmentsin adapting the electrolyte solution of the present invention todifferent gel chemistry and keeping a proper pH ratio compatible withgel and buffer. Preferably the concentration of Tris is from about 50 mMto about 150 mM, or from about 50 mM to about 250 mM and mostpreferably, at about 150 mM.

The ranges of concentration over which Tris base may be used forimproving the shelf life of gels prepared according to the presentinvention is from about 50 mM to about 375 mM, and preferably 150 mM.

Sodium Dodecyl Sulfate or Other Anionic Surfactants

Sodium dodecyl sulfate (SDS) (C₁₂H₂₅SO₄Na) is an anionic surfactant iscommonly used in preparing proteins for electrophoresis in the SDS-PAGEtechnique. The molecule has a tail of 12 carbon atoms, attached to asulfate group, giving the molecule the amphiphilic properties requiredof a detergent.

SDS may be optionally added to the electrolyte solution of the presentinvention. The superior results obtained with the electrolyte solutionsaccording to the present invention are obtained independently of thepresence of SDS. The ranges of concentration over which SDS may be usedfor the preparation of electrolyte solutions according to the presentinvention, for all the applications discussed herein are from 0.5% orless. Preferably, from about 0.1% or less, and most preferably, at 0.1%.

Other anionic surfactants may be included in the electrolyte solution ofthe present invention, such as, in a non-limiting manner facultative,one anionic surfactant to provide to the buffer denaturing propertiesfor protein analysis: SDS, sodium dodecyl sulphate, sodium laurylsulfate (SLS), sodium laurilsulfate or sodium, NaDS. The ranges ofconcentration over which they may be used for the preparation ofelectrolyte solutions according to the present invention, for all theapplications discussed herein are from 1.0% or less. Preferably, fromabout 0.1% or less, and most preferably, at about 0.1%.

Chelating Agents

Ethylenediaminetetraacetate (EDTA) has a role as a chelating agent, i.e.its ability to “sequester” metal ions such as Ca²⁺ and Fe³⁺. After beingbound by EDTA, metal ions remain in solution but exhibit diminishedreactivity. Other chelating agents may include ethylene glycoltetraacetic acid (EGTA), trisodium nitrilotriacetate, hydroxyethylethylenediamine trisodium acetate (trisodium HEDTA), diethylene triaminopentasodium acetate or uramil disodium acetate.

EDTA may be optionally added to the electrolyte solution of the presentinvention. The ranges of concentration over which EDTA may be used forthe preparation of electrolyte solutions according to the presentinvention, for all the applications discussed herein are from 0.5% orless. Preferably, from about 0.05% or less, and most preferably, at0.03%.

Methanol

In electrophoresis for transfer onto membranes, methanol is normallyadded to the transfer buffer to slow the protein migration and normallypermit a more equal protein transfer between the high molecular weightproteins and the low molecular weight. In standard protocol, it is usedas 10% or 20% of methanol. The efficacy of fast transfer withelectrolyte solutions according to the present invention containing 0%to 20% methanol is working equally well to 100% efficacy in transferunder all these conditions.

Use of the Electrolyte Solutions

In use the electrolyte solutions of the present invention is compatiblewith a very wide range of other buffer systems. The electrolyte solutionof the present invention may be used to run electrophoresis of gels ofany type, prepared with the same or with different buffer systems thanthat of the present invention, even including gels using differentchemistries, such as MOPS as a buffer (such as those described in USPatent publication No. 20060118418), under the appropriate conditions.

According to one embodiment, electrolyte solution according to thepresent invention containing Tris and the zwitterions EPPS (e.g. fromabout 10 mM to about 100 mM), ACES (e.g. from about 10 mM to about 75mM), MOPS (e.g. from about 10 mM to about 50 mM) and PIPES (e.g. fromabout 1 mM to about 10 mM) may be used to improve resolution andmigration speed of gels based on different chemistries than Leammli's.An anionic surfactant (such as SDS from about 0% to about 1%) may beincluded in such buffers to provide denaturing properties. Theresolution may be improved by improving the definition (or sharpness),by providing thinner or narrow bands) of the molecules separated, byimproving the separation between the molecules (i.e. the distancebetween the individual bands, or by both improving the definition (orsharpness) and separation of the molecules.

According to the present invention, electrophoresis includes theseparation of samples of DNA or protein or any other type of moleculethat may be separated accordingly, as well as their transfer ontomembranes or other suitable solid support such has nitrocellulose,nylon, PVDF or other types of membranes that are commonly used forapplications such as Western transfer and blotting.

The electrolyte solution according to the present invention may be usedin transfer systems of commercial make (e.g. wet, semi-dry or drytransfer systems). For examples, the electrolyte solution according tothe present invention may be employed in semi-dry or dry transfersystems such as Invitrogen XCell II™ Blot Module, Pierce™ Fast WesternSystem, Bio-Rad's Trans-Blot SD™ Semi-Dry Electrophoretic Transfer Celland equivalent equipment. Liquid (i.e. wet) systems such as Bio-Rad MiniTrans-Blot® Module, GE MiniVe Blot module and other equivalents.Electrophoresis systems such as the eSTain™ Protein Staining System fromGenScript, which comprise both the electrophoresis unit and the powersupply may also work with the electrolyte solution according to thepresent invention. Other equivalent systems also functions as well.

The electrolyte solution of the present invention may be used as thebuffer system in most of gels used in molecular biology andbiochemistry, as described in classical references such as: Uriel 1966,Bull. Soc. Chem. Biol. 48:969; Peacock & Dingman 1967, Biochem 6(6),1818-1827; Peacock & Dingman 1968, Biochem 7(2), 668-674; Gaal,Electrophoresis in the separation of biological macromolecules, p 422,Wiley, 1980. The electrolyte solutions of the present invention may beincluded in acrylamide gels (polyacrylamide gels), under native (withoutSDS) or denaturing conditions (with SDS) that are typically preparedwith acrylamide concentrations from about 4% to about 25%. Theelectrolyte solutions of the present invention may be included inagarose gels that are typically prepared with agarose concentrationsfrom about 0.5% to about 3%.

EPPS (or HEPPS), TAPS, TES, BES or Glygly can be added to the Laemmlibuffer Tris-Glycine-SDS at a working concentration of 25 mM to 150 mMand preferably at 50 mM and accelerate the migration efficacy of theLaemmli buffer by about 30%. In electrophoresis for transfer ontomembranes, methanol is normally added to the transfer buffer to slow theprotein migration and normally permit a more equal protein transferbetween the high molecular weight proteins and the low molecular weight.In standard protocol, it is used as 10% or 20% of methanol. The efficacyof fast transfer with electrolyte solutions according to the presentinvention containing 0% to 20% methanol is working equally well to 100%efficacy in transfer under all these conditions.

ALTERNATIVE EMBODIMENTS Example I

Exemplary electrolyte solutions compositions were prepared to be usedwith polyacrylamide or agarose gels made using classic recipes such asthe Laemmli buffer system (Tris-Glycine-SDS). The exemplary electrolytesolutions were prepared and tested using a common basic recipe:

Zwitterions, 50 mM, Tris (Base), 100 mM, SDS 0.1%, and EDTA 0.03%. Foreach prepared solution, the pH is measured, and if the value was outsideof the pH 7.9 to 8.6 range, it was adjusted with HCl to acidify, or NaOHto make more basic.

500 ml of running buffer were prepared for each zwitterions, and runagainst a precast IDGel™ (made with Tris pH 8.3 as buffering solution).After the electrophoretic run, the migration speed for the migrationfront to reach the bottom of the gel, and the resolution of a prestainedmolecular weight marker containing 10 bands weighing between 15 KDa to175 KDa was measured. The resolution of each of these bands has beencompared to a control IDGel™ ran with the standard Laemmli runningbuffer (Tris-Glycine-SDS). The results we obtained are as follows intable 1.

TABLE I Gel size (migration Running Zwitterion length) time ResolutionAMPD 60 mm 45 min ++ AMPSO 60 mm 50 min +++ BES 60 mm 40 min ++(+) CAPS60 mm 40 min ++ CAPSO 60 mm 60 min (+++) CHES 60 mm 42 min +(+) EPPS (orHEPPS) 60 mm 45 min +++ Glygly 60 mm 45 min ++(+) POPSO 60 mm 50 min ++TAPS 60 mm 35 min +++ TES 60 mm 40 min +++ TGS* 60 mm 60 min ++*Tris-Glycine-SDS = baseline

The resolution is compared by measuring the thickness of proteins bandsof 30 KDa, 21 KDA, 15 KDA and 14.3 KDa after migration in the test gel.A reduction of the band thickness by at least 50% will be noted as a“+++” sign. Thicker protein bands, indicative of lower resolution, isshown with less “+” signs. The baseline is “++”, which is the resultobtained by a Tris-Glycine-SDS gel, and a lesser number of “+” signsindicate thicker bands than baseline, and therefore worst averageresolution.

Now referring to FIG. 2. The change in the band thickness and resolutionof four low molecular weight proteins was measured. FIG. 2 shows thatthe presence of EPPS (or HEPPS) in the buffer helps improving theresolution by at least 50%. As well, the complete migration of each ofthese gels was accelerated by 30% using EPPS (or HEPPS).

Example 2

The same electrolyte solutions tested as running buffer above can beused to prepare the acrylamide or agarose gel composition and provideincreased resolution, but not necessarily separation speed.

TABLE 2 Gel size (migration Running Zwitterion length) time Resolution¹EPPS (or HEPPS) 60 mm 50 min +++ Glygly 60 mm 50 min ++ TGS* 60 mm 60min ++

Example 3

Some of these compounds have been identified to have a positive impacton stabilizing the gel matrix and allowing an extended storage, or morestable aging, providing longer shelf-life at 4° C. or room temperature.Now referring to FIG. 3. The figure shows that gels kept at 4° C. andtested every 30 days to detect the impact of aging. The results showthat after 360 days, the gel migration is similar to a fresh 1-day oldprecast gel. These results confirm the stability of the gel matrix whenEPPS (or HEPPS) is used as a buffering agent.

Example 4

The buffer of the present invention may also be used a transfer bufferin applications such as the transfer of proteins from acrylamide gels(Western transfer). Exemplary buffers were prepared according to table3, below.

TABLE 3 # Compound T0 T1 T2 T3 T4 Tris (g) 3 18 3 9 6 Glycine (g) 14.4 —— — — EPPS(or — 12 2 6 4 HEPPS) (g) H₂O (ml) to 800 800 800 800 800Methanol (ml) 200 200 200 200 200 T0 = Laemmli buffer with a glycinebase.

Transfer of protein markers from a precast IDGel™ (made with Tris pH 8.3as buffering solution). The transfer was most efficient with the recipeof buffer T4.

Example 5

Several buffers according to the present invention are tested. Thezwitterions tested are listed in table 4

TABLE 4 Testing of zwitterions of interest Composants Symetrical CathodeAnode Tris (g) 6 6 1 EPPS (or 4 1 4 HEPPS) (g) Gly-Gly (g) 4 1 4 CAPSO(g) 4 1 4 BES (g) 4 1 4 Methanol 0% 10% 20% 0% 10% 20% 0% 10% 20% Totalvolume 1 litre 1 litre 1 litre

The transfer efficacy of the different tested zwitterions are:

EPPS 100% (HEPPS) Gly-Gly 100% CAPSO 90% BES 80%

Example 6

Several buffers according to the present invention are tested in anasymmetric configuration, as indicated in table 5.

TABLE 5 transfer of asymmetric buffer: Components Cathode Anode Tris (g)6 1 Gly-Gly (g) or EPPS 1 6 (or HEPPS) Methanol 10% 10%

In each case, for the presented asymmetric arrangements with Gly-gly orEPPS (or HEPPS) buffer, the transfer is 100% efficient and occurs in 15min of electrophoresis.

Example 7

The Tris/Gly-Gly buffer according to the present invention (see Example5) may be used for Western transfer without any further modification.The buffer also supports an accelerated separation/transfer: a currentof 275 mA results in a complete transfer in 15 minutes or less. Methanolis normally added to the transfer buffer to slow the protein migrationand normally permit a more equal protein transfer between the highmolecular weight proteins and the low molecular weight. In standardprotocol, it is used as 10% or 20% of methanol. The efficacy of fasttransfer with electrolyte solutions according to the present inventioncontaining 0% to 20% methanol is working equally well to 100% efficacyin transfer under all these conditions.

Example 8

To perform semi-dry and/or wet transfer, the an electrolyte solutionaccording to the present invention may be used. The solution containingEPPS (or HEPPS) is prepared as a 0.3× solution: 6 g Tris, 4 g EPPS (orHEPPS) in a total volume of 1 L of distilled water. The electrolytesolution may comprise between 0% to 20% methanol, and preferably between10 and 20% methanol to improve transfer of large molecular weightproteins. The transfer of sample from the gel at 275 mA is typicallycompleted in 10 to 15 minutes.

Example 9

For use in electrophoresis transfer system such as the eStain™, sincethe volume of electrolyte solution required is small, two blottingpapers are soaked in a 5× electrolyte solution according to the presentinvention (90 g Tris, 60 g EPPS (or HEPPS) and up to a 1 L total volumewith water) and are placed on each side of the gel from which theproteins are transferred. The transfer is complete in 10 minutes atabout 80 mA to about 300 mA, 100% of the sample is transferred to thePVDF membrane used.

Example 10 Improved Buffer for MOPS Based Gels

A protein electrophoresis running buffer that accelerates the separationspeed and increase resolution of MOPS based gels and other gels. Thebuffer is based on the Tris-EPPS-SDS buffer in combination with theACES, MOPS, and PIPES zwitterions This buffer will be advantageous ongels based on a different chemistry than Laemmli's, for example MOPSbased gels such as NuPage™ from Invitrogen and EZ-Run™ fromThermoFisher, chemistry proprietary to Amresco.

The basic recipe of the buffer is a combination ofTris+SDS+EPPS+ACES+MOPS+PIPES, at a pH 8.5 or less and may facultativelyinclude one anionic surfactant to provide to the buffer denaturingproperties for protein analysis (e.g. SDS, sodium dodecyl sulphate,Sodium lauryl sulfate (SLS), sodium laurilsulfate or sodium, NaDS).

The proportions of this electrolyte solution are as indicated in table6:

TABLE 6 Components of the composition mM % weight 1X TRIS 49.9% 74.3 SDS2.8% 3.5 EPPS 19.4% 27.7 ACES 9.7% 19.2 PIPES 1.7% 2.0 MOPS 16.6% 95.6

Under such proportion, the buffer has a pH of 7.4, which is compatiblewith the MOPS based gels having a pH of 7.0.

Example 11 Improved Buffer for MOPS Based Gels

This study was made using the liquid gel matrix EZ-Run™ fromThermoFisher™, a product developed by Amresco™. This liquid gel matrixhas the advantage to be stable over one year at room temperature andallow to quickly cast electrophoresis gels. The gel matrix comes withits specific running buffer, which has to be used with it. Therecommended running conditions are 150V.

The main problem related to this liquid gel matrix is that it takeslonger than a normal Laemmli gel or other precast gels available on themarket to make a normal gel separation. For this reason, the EPPS basedrunning buffer of the present invention is adapted to this MOPS basedgel matrix (see US Patent publication No. 20060118418).

The performances of the new buffer are represented in the followingtable. “Long gel”=10×10 cm and “Short gel”=10×8.2 cm.

TABLE 7 Performance EZ-Run vs IDFast Univ Long gel 150 V EZRun 110 min EPPS mix 80 min 27% faster Long gel 200 V EZRun 80 min EPPS mix 50 min38% faster Short gel 150 V EZRun 65 min EPPS mix 45 min 31% faster Shortgel 200 V EZRun 50 min EPPS mix pH 7.4 35 min 30% faster EPPS mix 2 pH 830 min 40% faster Also compatible with Laemmli gels (short gels, 200 V)EZRun 50 min EPPS only buffer 35 min EPPS mix 29 min 17% faster EPPS mixpH 7.4 29 min

The gel prepared with the EZ-Run gel matrix and migrated with the EZ-Runspecific running buffer, when compared to the EPPS based buffer of thepresent invention, as described in Example 9, clearly shows an advantagein migration speed for the electrolyte solution of the present inventionwithout loss of resolution (See. FIG. 6).

The pH of the electrolyte solution will also affect the speed. TheEZ-Run gel matrix has a pH of 7.0. The EZ-Run running buffer has a pH of8.0. The electrolyte solution of the present invention (according toExample 9) has a pH of 7.4 once mixed, but adjusting it to 8.0 with NaOHhas an important impact on the speed.

The electrolyte solution of the present invention can be used alone, aswell as in combination with the buffer specifically designed for thisgel matrix (EZ-Run running buffer in this example). The proportion canchange as desired, and shows compatibility in the resolution and aproportional speed of migration. Based on the results above, it appearsthat the EZ-Run buffer slows down the migration.

Example 12 Improved Resolution and Gradient Effect of EPPS Based Gels

An electrolyte solution according to the present invention is identifiedto have a positive impact on the resolution of the protein bandsseparated on the gel. The electrolyte solution improves both thedefinition (sharpness) and the separation of the protein bandsseparated. Now referring to FIGS. 7A, B and C, three polyacrylamide gelswere prepared. Gel A is a precast IDGel™ Tris-Glycine gel with 10%acrylamide; gel B is an IDGel™ Tris-Glycine gel with a gradient of 4% to20% acrylamide; gel C is a gel containing 50 mM EPPS with 10%acrylamide, according to the present invention. All gels were run in a50 mM EPPS and 50 mM Tris containing buffer (IDFast) according to thepresent invention. FIG. 7 shows that compared to the Tris-glycine gels,the resolution of the EPPS gel is much improved. The band linked by aline corresponds to 25 kDa.

For example the EPPS containing gel (FIG. 7C) is capable of resolvingproteins better than a Tris-glycine gel of identical acrylamidepercentage (compare FIGS. 7A and C, lanes 1 vs. 9, lanes 2 vs. 10, andlanes 3 vs. 8). It may be observed that the smaller molecular weightproteins (e.g. 6 kDa in lanes 3 and 8, 10 kDa and 17 kDa in lanes 1 and9 and 19 kDa and 15 kDa in lanes 2 and 10) are well distanced anddistinguishable on EPPS containing gel, while they are poorly separatedand fused with the dye front in the Tris-glycine gel. Furthermore,higher molecular weight bands are somewhat sharper.

Also, the EPPS containing gel (FIG. 7C) is also capable of providingbetter resolution than a Tris-glycine gradient (4 to 20% acrylamide) gel(FIG. 7B). The gradient gel of FIG. 7B provides distinctly betterresolution than that of the Tris-glycine 10% gel, providing separationof the smaller molecular weight proteins (compare lanes 1 and 5, 2 and6, and 3 to 4 and 7). Nevertheless, when compared to the gradient gel,the EPPS containg gel (compare FIGS. 7B and C) provides for greaterdistance between the protein bands across the entire range of molecularweight, especially for the smaller molecular weight proteins. The EPPScontaining gel permits for small differences in molecular weight ofprotein to be made (e.g. compare lanes 4-5 to lanes 8-9. Interestingly,the EPPS containing gel appears to migrate in a similar “gradient-like”manner that is more commonly observed in gradient gels such as in FIG.7B, this without the presence of a gradient in the EPPS containing gel,which is of a fixed 10% acrylamide concentration. Note that the dyemigration front of the EPPS containing gel (FIG. 7C) has already exitedthe gel, while it remains present in the other gels.

The embodiments and examples presented herein are illustrative of thegeneral nature of the subject matter claimed and are not limiting. Itwill be understood by those skilled in the art how these embodiments canbe readily modified and/or adapted for various applications and invarious ways without departing from the spirit and scope of the subjectmatter disclosed claimed. The claims hereof are to be understood toinclude without limitation all alternative embodiments and equivalentsof the subject matter hereof. Phrases, words and terms employed hereinare illustrative and are not limiting. Where permissible by law, allreferences cited herein are incorporated by reference in their entirety.It will be appreciated that any aspects of the different embodimentsdisclosed herein may be combined in a range of possible alternativeembodiments, and alternative combinations of features, all of whichvaried combinations of features are to be understood to form a part ofthe subject matter claimed.

The invention claimed is:
 1. An electrophoresis gel comprising anelectrolyte solution comprising: Tris(hydroxymethyl)aminomethane (TRIS);at least one zwitterion chosen from 2-amino-2methyl-1,3-propanediol(AMPD), N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),2-(cyclohexylamino)ethanesulfonic acid (CHES),3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS),4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholinopropanesulfonic acid (MOPS),piperazine-N,N′-bis[2-hydroxypropanesulphonic]acid (POPSO),N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),(1,4-piperazinediethanesulphonic acid) (PIPES), andpiperazine-N,N′-bis(3-propanesulfonic Acid) (PIPPS); water; sodiumdodecyl sulfate (SDS); and methanol; wherein said electrolyte solutionis for accelerating, or improving resolution, or improving transferefficacy, or accelerating and improving resolution, or accelerating andimproving transfer efficacy of gel electrophoresis; wherein said gel ischosen from an acrylamide gel at a concentration from less than 1%(wt/vol) to about 25% (wt/vol) or an agarose gel at a concentration fromabout 0.5% (wt/vol) to about 3% (wt/vol); and wherein the pH of said gelis from about 8.0 to about 8.8.
 2. The electrophoresis gel of claim 1,wherein said methanol is up to 20%.
 3. The electrophoresis gel of claim1, wherein the concentration of Tris(hydroxymethyl)aminomethane (TRIS)is from about 10 mM to about 500 mM.
 4. The electrophoresis gel of claim1, wherein the concentration of the zwitterion is from about 1 mM toabout 500 mM.
 5. The electrophoresis gel of claim 1, wherein theconcentration of sodium dodecyl sulphate (SDS) is 0.5% (wt/vol) or less.6. The electrolyte solution according to claim 1, wherein saidzwitterions is a combination of4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),(2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES) and(1,4-piperazinediethanesulphonic acid) (PIPES).
 7. A method ofaccelerating or improving the resolution, or improving transferefficacy, or accelerating and improving the resolution, or acceleratingand improving transfer efficacy of the electrophoretic separation of atleast one sample comprising the step of: applying a voltage to anelectrophoresis gel according to claim 1 containing the at least onesample therein.
 8. The electrophoresis gel according to claim 1, furthercomprising a chelating agent chosen from ethylenediaminetetraacetate(EDTA), ethylene glycol tetraacetic acid (EGTA), trisodiumnitrilotriacetate, hydroxyethyl ethylenediamine trisodium acetate(trisodium HEDTA), diethylene triamino pentasodium acetate and uramildisodium acetate.
 9. The electrophoresis gel according to claim 8,wherein the concentration of ethylenediaminetetraacetate (EDTA) is 0.5%(wt/vol) or less.
 10. An electrophoresis gel comprising an electrolytesolution comprising: Tris(hydroxymethyl)aminomethane (TRIS); at leastone zwitterion chosen from 2-amino-2methyl-1,3-propanediol (AMPD),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),2-(cyclohexylamino)ethanesulfonic acid (CHES),3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS),4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholinopropanesulfonic acid (MOPS),piperazie-N,N′-bis[2-hydroxypropanesulphonic]acid (POPSO),N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),(1,4-piperazinediethanesulphonic acid) (PIPES), andpiperazine-N,N′-bis(3-propanesulfonic Acid) (PIPPS); water; sodiumdodecyl sulfate (SDS); and a chelating agent is chosen fromethylenediaminetetraacetate (EDTA), ethylene glycol tetraacetic acid(EGTA), trisodium nitrilotriacetate, hydroxyethyl ethylenediaminetrisodium acetate (trisodium HEDTA), diethylene triamino pentasodiumacetate and uramil disodium acetate; wherein said electrolyte solutionis for accelerating, or improving resolution, or improving transferefficacy, or accelerating and improving resolution, or accelerating andimproving transfer efficacy of gel electrophoresis; wherein said gel ischosen from an acrylamide gel at a concentration from less than 1%(wt/vol) to about 25% (wt/vol) or an agarose gel at a concentration fromabout 0.5% (wt/vol) to about 3% (wt/vol); and wherein the pH of said gelis from about 8.0 to about 8.8.
 11. The electrophoresis gel of claim 10,wherein the concentration of ethylenediaminetetraacetate (EDTA) is 0.5%(wt/vol) or less.
 12. The electrophoresis gel of claim 10, furthercomprising methanol.
 13. The electrophoresis gel of claim 12, whereinsaid methanol is up to 20%.
 14. The electrophoresis gel of claim 10,wherein the concentration of Tris(hydroxymethyl)aminomethane (TRIS) isfrom about 10 mM to about 500 mM.
 15. The electrophoresis gel of claim10, wherein the concentration of the zwitterion is from about 1 mM toabout 500 mM.
 16. The electrophoresis gel of claim 10, wherein theconcentration of sodium dodecyl sulphate (SDS) is 0.5% (wt/vol) or less.17. The electrophoresis gel according to claim 10, wherein saidzwitterions is a combination of4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),(2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES), and(1,4-piperazinediethanesulphonic acid) (PIPES).
 18. A method ofaccelerating or improving the resolution, or improving transferefficacy, or accelerating and improving the resolution, or acceleratingand improving transfer efficacy of the electrophoretic separation of atleast one sample comprising the step of: applying a voltage to anelectrophoresis gel according to claim 10 containing the at least onesample therein.
 19. An electrophoresis gel comprising an electrolytesolution comprising: Tris(hydroxymethyl)aminomethane (TRIS); at leastone zwitterion chosen from 2-amino-2methyl-1,3-propanediol (AMPD),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),2-(cyclohexylamino)ethanesulfonic acid (CHES),3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),N-(2- Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS),4-(N-Morpholino)butanesulfonic acid (MOBS), 3-N-Morpholinopropanesulfonic acid (MOPS),piperazie-N,N′-bis[2-hydroxypropanesulphonic]acid (POPSO),N-Tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), (2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES),(1,4-piperazinediethanesulphonic acid) (PIPES), andPiperazine-N,N′-bis(3-propanesulfonic Acid) (PIPPS); water; and sodiumdodecyl sulfate (SDS), wherein the concentration of said SDS is 0.5%(wt/vol) or less.
 20. The electrophoresis gel according to claim 19,further comprising methanol.
 21. The electrophoresis gel of claim 20,wherein said methanol is up to 20%.
 22. The electrophoresis gel of claim19, wherein the concentration of Tris(hydroxymethyl)aminomethane (TRIS)is from about 10 mM to about 500 mM.
 23. The electrophoresis gel ofclaim 19, wherein the concentration of the zwitterion is from about 1 mMto about 500 mM.
 24. The electrophoresis gel of claim 19, furthercomprising a chelating agent chosen from ethylenediaminetetraacetate(EDTA), ethylene glycol tetraacetic acid (EGTA), trisodiumnitrilotriacetate, hydroxyethyl ethylenediamine trisodium acetate(trisodium HEDTA), diethylene triamino pentasodium acetate and uramildisodium acetate.
 25. The electrophoresis gel according to claim 24,wherein the concentration of ethylenediaminetetraacetate (EDTA) is 0.5%(wt/vol) or less.
 26. The electrophoresis gel according to claim 19,wherein said zwitterions is a combination of4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),(2-[2-acetamino]-2-aminoethanesulphonic acid) (ACES), and(1,4-piperazinediethanesulphonic acid) (PIPES).
 27. A method ofaccelerating or improving the resolution, or improving transferefficacy, or accelerating and improving the resolution, or acceleratingand improving transfer efficacy of the electrophoretic separation of atleast one sample comprising the step of: applying a voltage to anelectrophoresis gel according to claim 19 containing the at least onesample therein.